Method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair

ABSTRACT

A medical composition is disclosed, which is injectable and which comprises a mixture of native components, which are obtainable by culturing one or more cell samples from a human or animal during normal conditions, said native components being included in the group consisting of growth factors, extracellular matrix proteins, and other substances produced by said cell samples during normal conditions, and a pharmaceutically acceptable carrier, as well as a method for producing the native components, a method for producing the medical composition, a method for treating a subject in need of tissue repair by injection of the medical composition, and use of said mixture of native components for the production of said medical composition for tissue repair via injection. A medical composition is disclosed, which is injectable and which comprises a mixture of native components, which are obtainable by culturing one or more cell samples from a human or animal during normal conditions, said native components being included in the group consisting of growth factors, extracellular matrix proteins, and other substances produced by said cell samples during normal conditions, and a pharmaceutically acceptable carrier, as well as a method for producing the native components, a method for producing the medical composition, a method for treating a subject in need of tissue repair by injection of the medical composition, and use of said mixture of native components for the production of said medical composition for tissue repair via injection.

This is a division of application Ser. No. 12/443,282, filed Dec. 23,2009, which is a § 371 National Stage of International Application No.PCT/SE2007/000875, filed Oct. 2, 2007, which claims benefit ofprovisional Application No. 60/848,399 filed Oct. 2, 2006, and claimsforeign priority to SE Application No. 0602109-1, filed Oct. 2, 2006,all of which are incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a medical composition, to a method forproducing a mixture of native components, to a method for producing saidmedical composition, to a method for treating a subject in need oftissue repair, and to the use of said mixture of native components forthe production of said medical composition for tissue repair viainjection.

BACKGROUND ART

Replacing or repairing damaged or lost tissue is among the mostexpensive medical therapies and cost billions of dollars a year all overthe world. There is an increasing demand for new methods and materialsthat can be applied in tissue engineering.

The experience of transplanting multiplied stem cells or specific donorstem cells into the human body, e.g. in joints, heart, brain andendocrine organs, has not been successful in showing significant longterm survival of the transplanted cells. In a few studies, however, someimpact on vascular ingrowth and repair with cell recruitment and minorclinical improvement has been reported.

The most obvious reason for the reported short term effect oftransplanted stem cells on local repair (Gazit et al. 2006) is in ouropinion that the transplanted cells during their short survival time actas a local cell factory producing a cascade of growth factors andextracellular matrix proteins capable of recruiting progenitor stemcells. Recent studies using colony-stimulating factors to enhance localtissue generation recruiting existing local or circulating stem cellsstrengthen this hypothesis. It has been demonstrated that the humanbrain contains cells with stem cell-like properties and an ability togenerate new neurons from generator stem cells (Brederlau et al 2004).It has also recently been shown that the combination of transplantingneuroprecursor cells together with single growth factors in injured ratspinal cord in an acute phase will slightly improve the nerve function.The same effect was not seen if the transplantation was carried outafter 8 weeks (Karima et al. 2006).

Bio-engineered tissue has been successfully used for replacementpurposes in a limited number of clinical applications for example in thetreatment of bone defects, diabetic ulcers and for tendon ruptures. Themost successful approach has been to select different cell types thatexhibit the function and characteristics of the tissue of interest. Thebest long term results have been reported in the knee for isolatedchondral defects using autografts, i.e. chondrocytes cultured in abioreactor on a scaffold using, for example, a 3-dimensional matrix, acollagen fleece or hyaluronan. However, mincing cartilage anddistributing it on a similar matrix in a one-stage procedure withoutprior culturing gives as good results as with autologous chondrocyteimplantation. In both cases a hyaline-like cartilage will be the result.The implant will often integrate poorly with neighbouring cartilage.This almost always gives a scar tissue leaving a cleft, in the areabetween healthy cartilage and the transplant (Yiling et al 2006).

In WO2002/067762 A2 a muscle polymer construct for bone tissueengineering is described, wherein a bone grafting material comprising apolymer scaffold loaded with bone morphogenetic proteins and populatedwith muscle cells is prepared to synthesize bone tissue.

In US20050019419 a tissue graft composition comprising liver basementmembrane and a method of preparation of this tissue graft compositionare described. The graft composition can be implanted to replace orinduce the repair of damaged or diseased tissues.

U.S. Pat. No. 6,096,347 describes the use of submucosal tissue of awarm-blooded vertebrate to manufacture a tissue graft composition thatinduces the formation of endogenous cardiac tissues in vivo upon contactof the cardiac tissues with the manufactured composition.

WO 01/14527 refers to a conditioned medium composition containing skinagents produced from cultured cells of skin and a carrier agent fortopical application on the skin.

US 2002/0049422 A1 discloses a topical composition comprising differentgrowth factors.

WO 02/24219 refers to an isolated protein complex comprising a growthfactor binding protein, vitronectin and a growth factor, as well as asurgical implant and a skin regeneration medicament comprising saidcomplex.

In view of the prior art there still remains a need to improve thereplacing and repairing of damaged or lost tissue within the body ofhuman and animal subjects. The applications of repairing and replacingdamaged or lost tissue are extensive and the conditions to be treatedare many.

As will be apparent from the following, the present invention isdirected to solving such needs.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a medical compositionwhich is injectable and which comprises a mixture of native components,which are obtainable by culturing one or more cell samples from a humanor animal during normal conditions, said native components beingincluded in the group consisting of growth factors, extracellular matrixproteins, and other substances produced by said cell samples duringnormal conditions, and a pharmaceutically acceptable carrier.

The present invention relates in a further aspect to a method forproducing a mixture of native components comprising the steps of:

-   -   adding one or several different cell samples of human or animal        origin to a bioreactor containing a nutrient medium, wherein a        culturing medium is obtained;    -   cell culturing during normal conditions;    -   drawing off at least once the culturing medium including the        native components produced during the cell culturing;    -   separating the native components from the culturing medium        thereby obtaining a mixture of native components.

The present invention relates in still a further aspect to a method forproducing the medical composition, wherein a carrier is added to themixture of native components produced, before or after an optionalfreeze-drying step.

The present invention relates in another aspect to a method for treatinga human or animal subject in need of tissue repair by injection of saidmedical composition.

The present invention relates in still another aspect to use of saidmixture of native components for the production of said medicalcomposition for tissue repair via injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of the method according to the invention,wherein cells are taken from a certain kind of tissue and thereafter arecultured in one or more bioreactors. The native components produced,such as growth factors and an extracellular matrix proteins (GECM), arethen combined with a chemically and mechanically optimized carrier,wherein a medical composition is formed. This is then injected into thehost tissue where the cells are recruited and proliferation anddifferentiation are enhanced.

FIG. 2 depicts a carrier with different modules of elasticity beingsoaked or preloaded with the native components, such as GECM producedaccording to the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In one embodiment, the method according to the invention furthercomprises freeze-drying of the separated native components such as GECM.Any conventional freeze-drying method known to a person skilled in theart may be used.

According to the present invention, each cell sample is or originatesfrom a tissue biopsy from a human or animal subject. Examples of animalsubjects from which the tissue biopsy can be taken are preferably, butnot limited to, pigs, cows, and goat. The tissue biopsy is preferablytaken from, but is not limited to, one or more of the following tissues:muscle, fat, cartilage, skin, nerves, liver, bone, and/or teeth.

In the present context, the wording “tissue biopsy” is meant to be abiopsy taken from any tissue in the body directly by removing thedesired sample and adding the sample directly to the bioreactor.Preferably, the tissue biopsy may be minced, enzymatically degraded,treated or enriched before adding it to the bioreactor.

In the present context, the wording “cell sample of human or animalorigin” is meant to mean a cell sample taken from a human or animal atany developmental stage, i.e. any stage from fertilization and onwards,e.g. a cell sample taken from the embryonic stage or before theembryonic stage or at any other developmental stage. The cells to becultured could be target-specific for, for example, a joint, elasticcartilage in the ear, nerves, hip abductor muscles, tendons from fascialata, skin from the upper arm, and mesenchymal cells from the iliaccrest etc.

In the present context, the wording “one or several different cellsamples” means that one specific cell sample from a certain tissue maybe used in the subsequent cell culturing step. More than one cell samplemay also be used, i.e. two or more cell samples from different tissues,for instance one cell sample from muscle and one cell sample fromcartilage etc, but only in specific situations or when the differenttissues have additional stimulatory effects.

In the present context, the wording “bioreactor” is meant to be aconventional bioreactor available for culturing of cells. A personskilled in the art may easily select a known reactor to be used in themethod of the invention. In one embodiment, use is made of a bioreactorwith two or more interconnected chambers separated by a millipore filterwhich allows for the native components produced, such as growth factorsand extracellular matrix proteins (ECM), to diffuse into the secondchamber or third chamber and so on. At predetermined intervals theculturing medium is drawn off and the native components separated andfreeze-dried under sterile conditions.

The native components produced may be sterilized by use of any one ofseveral sterilization techniques, e.g. by β or γ radiation at least 1.2MRad; by sterile filtration, wherein the whole process including thefreeze-drying and packaging is performed in a sterile bench; or by useof gassing with ethylene oxide (ETO). During the gassing operation thepackage, e.g. a bag, in which the treated medical product is placed forstorage after freeze-drying, has to be degassed during several weeks,normally about 4-6 weeks, wherein the ethylene oxide inside the packageslowly leaks out from the walls of the package.

In the present context, the wording “culturing medium” should bedistinguished from “nutrient medium”. The nutrient medium is the freshmedium with all the necessary nutrients required for growth of thecells, before the cells have been added. Once the cells have been addedto the nutrient medium the medium will be regarded as a culturing mediumwith the cells included as well as the native components produced duringcell culturing. When drawing off the culturing medium including thenative components the cells are usually not present in the culturingmedium as they have already been separated from the culturing medium inthe bioreactor with the two interconnected chambers. However, it ispossible that parts of the cells and a small number of cells areincluded in the culturing medium after filtration. Alternatively, inanother embodiment, the cells are included in the culturing medium whendrawn off from the bioreactor and are thereafter separated from theculturing medium by any conventional means.

In the present context, the wording “nutrient medium” means a nutrientmedium as used in its conventional context, i.e. a medium such as DMEMor specific growth media suitable for different cells and cell lines,which allows for the growth of cells and for the production of thenative components. The nutrient medium should provide certainconditions, such as hormones or growth factors that usually occur invivo. The choice of nutrient medium is dependent on the desireddirection of cell growth and the cells used. Substances that may bepresent in the nutrient medium are growth factors, nutrients such asglucose or other sugars as needed. Other substances that may be includedin the nutrient medium are, for instance, antibiotics.

In the present context, the word “normal conditions” is used to definethe environment in which the cells proliferate and stay viable in thesame or essentially the same manner as in their natural environment suchas in the human or animal body. A person skilled in the art is familiarwith what is meant by normal conditions and is able to arrange for thisduring culturing.

In another embodiment, the cell sample is a specific cell line. The cellline can be commercially available. Any known cell line that iscommercially available can be used according to the invention and aperson skilled in the art can easily select any cell line suitable forproducing the native components as required by the ultimate application.The advantage of using a commercially available cell line is that themany different native components of different origin can be produced andstored. When a patient needs immediate tissue replacement or reparationdue to damaged or lost tissue, such as cardiac infarction or cerebralhaemorrhage, the stored native components produced by the commerciallyavailable cell lines may be used directly by injecting the medicalcomposition comprising the native components with the desired carrier,if needed. In some instances, immediate access to the native componentsand the carrier is required within the first few days or weeks followingan acute emergency or injury. There are today commercial cell-lines thatcould be used for repetitive drawing off of the native components, thusmaking them available instantly in emergency situations. Specificmarkers, such as TGF-beta and IGF-1 and extracellular matrix proteins,could be used as marker proteins to establish the concentration. Inthese cases, the native components are of an allograft type, i.e. notthe patients own native components.

In the present context, the phrase “native components” is meant tocomprise all growth factors, all extracellular matrix proteins (ECM),and all further substances that are produced by the cells in theirnatural environment including native polymers and other proteins. In thepresent context, exactly the same components are produced in abioreactor as in the natural environment of the specific cell. Dependingon the tissues from which the cells to culture are taken, there areprobably hundreds of different proteins, biopolymers, and othersubstances and molecules that are produced. Extracellular matrixproteins are for instance, but are not limited to, fibronectin,vitronectin, chondroadherin and aggrecans.

In one embodiment, it is of interest to produce and isolate only growthfactors and extracellular matrix proteins (ECM). The combination ofthese is also called GECM (Growth factors and Extra Cellular Matrixproteins). These are the most important of the native components as theyprovide an optimal balance between downregulation and upregulation ofthe tissue regeneration process in which they are to be involved.However, in practice, all of the molecules defined by the expression“native components” are automatically present in the mixture or cocktailobtained and are effective for the subsequent regeneration processwithin the body, although highly satisfactory results also are obtainedwith only the GECM mixture.

A further embodiment of the invention is to provide a carrier, whichretain native components such as GECM and slowly releases them during aprolonged period of time (i.e. days, weeks or months) and which makesthe medical composition including the native components and the carrierinjectable into the human and animal body. As have been stated before,the carrier is selected according to the ultimate application, i.e. theorgan or place in the body where the carrier is to be injected.

In the present context, the term “native” means that the nativecomponents, of which several are proteineous, are in theirnon-denaturated state. Any chemical modification of the component isthus allowed as long as the biological activity is retained.

Examples of proteineous components are a parathyroid hormone, aprostaglandin (e.g. PGE₂), an osteoprotegrin (OPG), Indian hedgehogactivator, an NF-kappa B ligand (RANKL), a sex steroid, and a cytokine.

A growth factor is, in the present context, a general term for specificpeptides or proteins, which are released by certain cells and bind tospecific cell membrane receptor sites to influence cells to divide. Forexample, chondrocytes produce a number of growth factors, including, forinstance, transforming growth factor (TGF-β3), bone morphogenic protein(BMP-2), PTHrP, osteoprotegrin (OPG), Indian hedgehog activator, RANKL,basic fibroblast growth factor (bFGF) and insulin-like growth factor(IgF). The platelet-derived growth factor (PDGF) is a glycoprotein thatstimulates cell proliferation and chemotaxis in cartilage, bone, andmany other cell types after being produced by mesenchymal cells.Likewise, the basic fibroblast growth factor (bFGF) is produced locallyin bone during the initial phase of fracturing healing and is known tostimulate cartilage and bone-forming cells.

The super family of transforming growth factors (TGFs) is the mostextensively studied growth factor in the field of bone biology. Itcomprises an entire family of substances that includes the bonemorphogenetic proteins (BMPs). These are important cell-cell signallingsubstances, which induce cartilage and bone formation as well as promotethe differentiation of osteogenic precursor cells into osteoblasts. Itshould be noted that pure forms of BMPs, some produced by geneticengineering, are non-immunogenic and non-species-specific.

The growth factor can also be a fibroblast growth factor (FGF) or avascular endothelial growth factor.

A mixture of autologous growth factors, which is derived from the buffycoat of cells collected during surgery, is produced by Interpore CrossInternational Inc., Irvine, USA. These leukocytes are said to beespecially rich in TGF-β and PDGF. A cocktail of growth factors from abovine-derived bone morphogenetic protein extract is also produced byNeOsteo, Intermedics Orthopaedics, Denver, USA.

The amounts of different components in the medical composition dependson the cell and tissue origin of the native components produced as wellas on the final application intended, i.e. the kind of tissue or organto be repaired.

Normally, the proportion between the native components and the carrierin the final medical composition is about 1:10 on weight part basis, butthis proportion may vary somewhat.

Preferably, cells of human origin are used as “factories” for producingthe native components. A suitable mixture or cocktail of growth factors,obtained from cultured chondrocytes, can be up to for instance 100 kDain size or more, such as between about 70 kDa and 10 kDa, preferablybetween about 60 kDa and 20 kDa, more preferably between 50 kDa and 30kDa. The size of the components is naturally not limited to the abovesizes since these are only exemplifying.

The wordings “mixture of native components” and “cocktail of nativecomponents” used herein are intended to mean a set of all or a part ofthe native components as defined above produced from a certain cell,cell line or tissue, i.e. all of the components produced in theirnatural environment. If desired, some of said native components could beremoved from the medium after culturing with a view to maintaining onlythe most important native component for the subsequent application, e.g.growth factors and extracellular matrix proteins (GECM). As statedabove, native components originating from different cells, cell lines,and tissues may be present in the same mixture or cocktail of nativecomponents. Such mixtures are also intended to fall within the scope ofthe expression “mixture or cocktail of native components”.

In one embodiment of the invention, the cell culturing is carried outunder rotating conditions. The rotation causes a better nutritionalflow, which makes the cells proliferate and produce GECM better. Therotation can be implemented by any conventional rotating means used inconventional cell culturing. In certain embodiments, the rotation maynaturally be excluded. It is further suitable, but not necessary, that athree-dimensional matrix is present under the cell culturing in order toincrease the production of the native components. It is beneficial ifthe matrix is spherical, for instance starch beads, which have aninterconnected pore system for dynamic nutritional flow. The matrix canbe chosen from, but is not limited to, beads, starch beads, polymerbeads, and beads of alginate, collagen, hyaluronic acid, and chitosan.Any known matrices such as three-dimensional matrices that are suitablefor use in cell culturing may naturally be used in accordance with theinvention. A person skilled in the art realizes what kind of matricescan be used for beneficial growth.

In one embodiment of the invention, the drawing off of the culturingmedium including native components takes place continuously and thedrawn-off culturing medium is replaced with new nutrient medium. In oneembodiment, two interconnected chambers separated by a millipore filtercan be used for cell culturing, which allows for the native components,e.g. growth factors and ECM protein molecules, to diffuse into thesecond chamber. At pre-determined intervals the culture media could bedrawn off and the native components separated and freeze-dried understerile conditions, see FIG. 1. The cell culturing is carried out duringfor at least 7 weeks, preferably at least 5 weeks, more preferably atleast 3 weeks, and most preferably at least 1 week, or less, e.g. 1, 2,3, 4, 5, or 6 days, before the drawing-off step is performed.

In one embodiment of the invention, the native components produced areseparated from the culturing medium by their size or weight or byaffinity, e.g. by centrifugation or are separated in a column.

After separation the separated single native components may be combinedin a mixture or cocktail before freeze-drying, as described above.Alternatively, each of the separated native components is furtherenriched before freeze-drying. After freeze-drying, the freeze-driedmixture or cocktail may be stored in a bag until use. As stated above,it is also possible to enrich any of the separated components, combinethem in a cocktail and then freeze-dry the same. Thus, it is possible toobtain one or more native components from biopsies of different origin(i.e. from different tissues) and then combine the obtained componentsafter culturing in a novel cocktail and then optionally freeze-dry thecocktail.

According to the present invention, a carrier is added to the separatednative components before or after freeze-drying. In a furtherembodiment, freeze-drying is not necessary, e.g. in acute situations inwhich an injection is to be made immediately. Thus, it depends on theultimate application whether a carrier is to be included or not beforefreeze-drying. The carrier has to be injectable into the human andanimal body when mixed with the native components, and is chosen in viewof the ultimate application, otherwise the body of the human or animalpatient could have difficulties accepting the native component carrierproduct. The term “injectable” used herein means that the medicalcomposition, i.e. the mixture of native components and carrier, and anyfurther pharmaceutically acceptable auxiliary components, e.g.antibiotics and rheology enhancing components needed, to be introducedinto the human or animal body can be injected into the body through aneedle or tube having an inner diameter of 0.2-6 mm, preferably 0.4-2mm. This means that such parameters as viscosity and carrier materialsize of the medical composition according to the present invention mustbe optimized for fulfilling this purpose, which is familiar to a personskilled in the art. Certain carriers are only suitable for certainapplications, which is obvious to a person skilled in the art. Thecarrier must of course be acceptable by the human and animal body and ischosen from the group consisting of natural or synthetic polymers, andceramic materials which are well-known in the art to be injectable. Thecarrier may be chosen from, but is not limited to, hyaluronic acid,fibrin glue, chitosan, dextran, collagen, alginate, and biopolymers ofdifferent materials having different modules of elasticity. Thebiopolymer of different materials having different modules of elasticitymay be prepared by welding/binding together two different materials, forexample a metal and a plastic, or a natural and synthetic polymericmaterial. Other materials that can be used in this biopolymer are, forinstance, any organic and inorganic materials. It is also possible thatthe biopolymer is a biopolymer with an elasticity gradient within thesame material.

In one embodiment of the invention the development of modifications ofnatural polymers, synthetic polymers and/or ceramic materials withdifferent elastic and viscoelastic properties will be prepared, see FIG.2. The carriers could be soaked or pre-loaded or fabricated withprepared native components such as GECM and/or combined with otherfactors such as biphosphonates or antibiotics and eventually be combinedwith the systemic application of colony stimulating factors or drugs toenhance recruitment of circulating progenitor cells and cell bindingproteins.

In one embodiment, the mixture of native components may be injecteddirectly, i.e. without having been mixed with any carrier, into or closeto a carrier, e.g. an implant, which is already present in the body atthe site at which the tissue regeneration is to take place.

The native component(s) produced according to the method of theinvention, when ultimately administered to the subject, are of anautograft or allograft character or both. The advantages of autograftsto a patient are realized since these are the patients' own nativecomponents that are used, whereby for instance rejection is avoided.Other problems that are avoided are infections due to different virusesthat are transferred to the host, even if the transferred implant issterilized. Thus, virus infections such as HIV and infections caused byprions or any other species can be avoided when the implant is of anautograft type.

The tissue repair could, for instance, take place in, but is not limitedthereto, joints, vascular parts, muscles, tendons, the spinal cord,nerves, bone tissue, fat tissue, heart, brain, and endocrine organs. Forinstance, the specific native components prepared could be injected tobiological carriers like hyaluronic acid to be used for joint injuries,fibrin glue for Achilles tendon injuries, bi-phasic α-tricalciumphosphate (injectable bone substitute) for vertebral fractures andspinal fusion, dextran for spinal cord injuries etc, provided that thecarrier has been implanted in advance at the place of the tissue repairprocess. Other areas where the native components could be used arestroke, Parkinson's disease and Alzheimer's disease using availableproven biological carriers. It is also possible to use the inventivenative components with a carrier as an implant for implantation to apatient who has had an organ, preferably an endocrine organ, or part ofan organ removed, and in order to provide a faster re-growth of theorgan tissue an implant (carrier including native components) asprovided herein is implanted to the patient.

In accordance with the present invention, a method for treating diseasessuch as, but not limited to, stroke, myocardial infarction, Parkinson'sdisease, Alzheimer's disease, cerebral haemorrhage, bone diseases,muscle diseases, peripheral nerve injuries and tendon disease, has beenprovided.

In a further embodiment a carrier, e.g. in the form of a polymer, may beadded to the reactor containing the cells to be cultured. The nativecomponents produced and brought to pass the filter(s) in the reactor maythen be recirculated back to the part of the reactor containing thecarrier and the cells to be cultured, wherein the native components arebound to the carrier. Such a carrier including the native components,e.g. growth factors and ECM proteins, may then be implanted directlyinto a tissue repair place in the human or animal body.

In still another embodiment, a carrier may be placed in a separatecontainer through which native components produced are brought to pass,wherein said native components are bound to said carrier, which then maybe implanted directly at a tissue repair site in the human or animalbody.

EXAMPLES

Preliminary studies (see details below) have been performed withchondrocytes as cell samples taken from six human knees at surgery,cultured in a bio-reactor with the culture media obtained and the GECMproduced separated from the cells at 3 weeks. Three differentconcentrations of the GECM cocktail were incorporated into an injectablematerial and implanted into rat abdominal muscle. A biphasic ceramicbone substitute was used as a carrier, and measurements with TGF-betaand IGF-1 as markers showed a clear dose response curve. Vascularingrowth and new bone could be demonstrated in the synthetic material ina muscle. This is only possible by the recruitment of circulating orlocal progenitor cell-stem cells that differentiated muscle cells intobone.

In another study (see details below) we have tested whether one type ofECM, chondroadherin, a non-collagenous cartilage protein with cellbinding properties could be used in the same ceramic bi-phasic bonesubstitute to induce bone.

It has been concluded earlier that chondroadherin may play a role inmaintaining bone cells on collagen matrices. We used anα-tricalciumphosphate and calcium sulphate bone substitute in an animalrabbit model with a well described bone harvest chamber. Thepharmacokinetics was studied showing a significant release ofchondroadherin and increase over the first two weeks. However, it wasnot possible to show any significant increase in new bone whether asapposition or growing into the material. Looking at the specificactivity counting for instance the number of osteoclasts on the materialagain, showed no difference to the plain bone substitute.

One could speculate that a different carrier or ECM would make adifference. However, it is more likely that either the concentration ofchondroadherin was too low to show any effect or that a combination ofextracellular matrix proteins and growth factors would be necessary tosignificantly increase the recruitment of circulating or localprogenitor cells to transform and induce bone.

Detailed Description of Preliminary Experiment 1

Growth Factor Cocktail

An autologous growth factor chondrocyte cocktail (AGFCC) was producedfrom ex vivo cultured, human cartilage cells. During arthroscopicexamination the surgeon took a biopsy from the non-weight-bearing areaof the proximal part of the medial or lateral condyle. The cartilagechip was 8 to 10 mm long and extending down to the subchondral boneplat. The biopsy contained roughly 200-300 mg of cartilage. The biopsymaterial was immediately placed in the nutrient medium and forwarded toa cell laboratory and placed in a special nutrient solution (DMEM F-12).

Production of Growth Factor Cocktail

The cell nutrient medium consisted of DMEM F-12. The cells were culturedin a medium for about 2 weeks, whereafter the culturing medium wascollected and saved as a 100×-concentrated portion. TGF-β and IGF-Iserved as marker proteins to establish the concentration of the growthfactor cocktail. The amount of TGF-β in the highest concentration of themedium was about 50-300 ng/ml and that of IGF-I was about 100-300 ng/ml.The cocktails were produced from 7 patients.

Mixture of Bone Substitute

The α-TCP with 20% CaS was mixed with the respective cocktails, the highconcentrated (100 times) cocktail was diluted 10 times and 100 timeswith 1% BSA-PBS. With the concentrations, the materials were divided byIBS-GF1 (high concentration), IBS-GF3 (low concentration). After mixingthe material was injected into a mould (5 mm in diameter×4 mm thick) andleft until set (12 h). The set pellets made from the injectable materialwere implanted in rat's muscles.

Animal Experiments

64 female Sprague-Dawley rats with body weights of 200-230 g were usedfor the experiment. The operation was performed under anaesthesia withchloral hydrate (300 mg/kg, B.W. intraperitoneally). The preparedspecimens made from the injectable material were implanted in abdominalmuscle pouches of rats. 8 rats in each group were killed at each of thefollowing time intervals; 3, 6, 12 and 24 weeks.

Specimen Preparation and Evaluation

The bone substitutes with surrounding muscles were harvested. The samplespecimens with tissue and materials were fixed in 4% formalin in buffer,decalcified and embedded in paraffin. They were cut into 5μ sections andstained with H&E staining. The histological analysis was done byobserving osteoblasts, mineralization and trabecular bone in and aroundthe materials. The size of new bone formation area was counted by usinga microscope with a computer imaging system (Kontron Bildanalyse,Germany). The score of bone induction was done by a score standard undermicroscope.

Single ECM Experiment, Chondroadherin

Biomaterial as carrier and chondroadherin as native component werecombined with the aid of a 2.5% aqueous di-sodium hydrogen phosphatesolution forming a paste. The paste was then distributed into moulds toform small cylindrical pellets each of 1 mm diameter and height. Duringin vitro tests, once dried, pellets were immersed in 100 microliters ofphosphate buffered saline solution and left to dissolve at roomtemperature. At periodic intervals ranging from 15 minutes to 2 weeks,the bathing solution was removed, centrifuged, and run through a UV-Visspectrophotometer (at 280 nm) to record absorbance readings. Eachsolution was then replaced back into the appropriate vial and thedissolution process allowed to continue. It was hoped that absorbancereadings would reflect the proportion and subsequent rate of release ofchondroadherin into the surrounding solution as pellets graduallydissolved.

During tests carried out in vivo, a single pellet made from theinjectable material was implanted bi-laterally into the proximal tibiaeof 6 rabbits with the aid of a bone chamber. One side contained a pelletwith incorporated chondroadherin while the other served as a control.Bone in-growth was then assessed via histological analysis followingharvest at intervals of 2 and 3 weeks. No bone ingrowth was observed.

The invention claimed is:
 1. A method for treatment of a human or animalsubject in need of tissue repair comprising preparing a medicalcomposition and injecting the medical composition into the human oranimal subject, wherein the medical composition comprises a mixture ofnative components included in the group consisting of growth factors,extracellular matrix proteins and other substances produced by cellsamples during normal conditions, wherein the steps of preparing themedical composition comprise: adding one or several different cellsamples of human or animal origin to a bioreactor containing a nutrientmedium, wherein a culturing medium is obtained; cell culturing duringnormal conditions, wherein the cell culturing is carried out underrotating condition; drawing off at least once the culturing mediumincluding native components produced during the cell culturing;separating the native components from the culturing medium therebyobtaining a mixture of native components, wherein the drawing off takesplace continuously and the drawn-off culturing medium is replaced withnew nutrient medium, said bioreactor having two or more interconnectedchambers separated by a Millipore filter which allows for the culturingmedium containing native components produced to diffuse into a secondchamber or third chamber and so on and for separation of the cells fromthe culturing medium containing native components in the bioreactor, andinjecting the medical composition into the human or animal subjectwherein said tissue repair takes place in joints, muscle, tendon, fattissue, or heart of the human or animal subject.
 2. The method accordingto claim 1, wherein the medical composition is of an autograft orallograft type or both.
 3. The method according to claim 1 for treatinga subject for myocardial infarction, muscle disease or tendon disease.